Toxicity of Flunixin Meglumine in Broiler Chickens

*#Muhammad Ramzan, *Muhammad Ashraf, **Khawaja Tahir Mahmood

*Department Of Pharmacology and Toxicology ,University of Veterinary and Animal Sciences, Lahore, **Sr .Pharmacist, DTL,Punjab, Lahore Pakistan.

#drramzan2004@yahoo.com

Abstract This study was designed to evaluate the toxicity of flunixin meglumine, a nonsteroidal anti-inflammatory drug which might serve as an alternate of diclofenac that has been shown to cause toxicity in vultures. Twenty five broiler chickens of five weeks of age were randomly divided into five equal groups and treated with flunixin meglumine at the dosage rate of 0, 1.25, 2.5, 5 and 10 mg/kg body weight for four consecutive days, intramuscularly. Clinical signs, mortality, gross and microscopic lesions in visceral organs were recorded and serum concentrations of uric acid, creatinine, AST, ALT and ALP were measured. The affected birds exhibited the signs of toxicity as anorexia, depression, lethargy and coma. Mortality rates in 2.5, 5 and 10 mg/kg groups were 20, 40 and 60%, respectively. Gross lesions included visceral gout characterized by multifocal to locally extensive urate precipitates on almost all the visceral organs, skeletal muscles and sub cutis. Microscopic lesions showed multifocal urate tophi frequently surrounded by inflammatory cells. A strong correlation in the toxicity of the drug and elevation of the serum uric acid and creatinine concentrations was observed in the affected birds. We propose that flunixin meglumine causes similar toxicity in birds as diclofenac does. Key words: Flunixin meglumine, Nonsteroidal anti-inflammatory drugs, Drug toxicity, Visceral gout, Renal damage, Broiler chickens

INTRODUCTION

Recently a sharp decline in vulture population has been observed in Southeast Asia (1, 2, 3). International Union of Conservation of Nature (IUCN) has estimated that at least three vulture species i.e. Gyps bengalensis, Gyps indicusand Gyps tenortuirus have become critically endangered (4). Many causes for decline in vulture population have been postulated including food shortage, habitat loss, human persecution, environmental contaminants and infectious agent (5). However, recent studies found that the residues of diclofenac, a non-steroidal anti-inflammatory drug (NSAID) may be responsible for the vulture mortalities (6). Diclofenac is an antipyretic, analgesic and anti-inflammatory drug and is commonly used in the region to treat sick domestic animals (7). Vultures consume diclofenac residues in the meat of dead animals treated with this drug shortly before death (6, 8, 9). Vultures play a significant role in maintaining the ecological balance so their presence in the ecosystem seems important. Therefore an alternate NSAID is needed to replace diclofenac in veterinary medicine which may, at least be equally efficacious but safer for the scavenging birds. Flunixin meglumine (FM) is another NSAID and is being used in veterinary practice around the world. Flunixin is used alone or in combination with antimicrobial agents to treat a variety of diseases in domestic animals like, lameness (10, 11), colic (12), endotoxemia (13), mastitis (14), metritis (15) and respiratory diseases (16). This suggests that flunixin may be a potential alternative of diclofenac for treatment of sick domestic animals.

Since it is hard to get vultures for research, in a preliminary study, we searched other bird species that exhibited diclofenac toxicity similar to that observed in vultures. The preliminary study showed that, similar to the observations in vultures, broiler chickens were also sensitive to diclofenac and suffered from toxicity (unpublished data). Moreover, other studies have also used chickens as a model to study NSAID’s pharmacological and toxicological effects (17, 18, 19). Therefore, the aim of this study was to evaluate whether flunixin can be used in animals as an alternative to diclofenac, without serious toxicity in vultures.

MATERIALS AND METHODS Birds: Twenty five, 1-day old broiler chicks were obtained from a local hatchery. The chicks were brooded on fresh litter. The room temperature was maintained at 33°C during first week and then reduced by 3° per week subsequently till 24°C and that temperature was maintained for rest of the period. Birds were vaccinated against ND, IBD and IB as per schedule and provided with clean water and commercial broiler starter ration ad libitum. Light remained on for 24 hours a day. Drug: A 50 mg/ml solution of flunixin meglumine (Loxin, Selmore Pharmaceutical, Pakistan) was used. Experimental Design: The birds were randomly divided into five groups A, B, C, D and E containing five birds in each group.

Muhammad Ramzan, et al /J. Pharm. Sci. & Res. Vol.4(2), 2012, 1748-1754

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Birds inphysiolodosage intramustreated w1.25, 2.5intramuspectoral alternatiof injectdiluting ClinicalExperimabnormaThe clinrecordednoted. Blood saBlood (1before odosing. glass tesamples BiochemSerum TransamAlkalineCreatinindiagnostSpectropCorporaUrate coWhite chwas anaconfirmawas conUrine urpositive Gross aThe birdwere nectreatmensacrificeat the recordedliver, hcollectedbufferedthrough sections stained standardexamine

n group A ogical norma

rate ofscularly. Birdwith flunixin5, 5 and 10 mscularly eve

muscles ively, for foution was mathe drug in d

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minase (ASTe Phosphatasne (CREA) tic kits (Rphotometer ation, Japan).onfirmationhalky materilyzed througation of uratnsidered posrate depositsand negativend histopathds that died cropsied immnt groups aned and examiend of ex

d for each bheart, spleend from eachd formalin.

paraffin embof 3µm th

with haemad procedureed for histopa

(control) wal saline solf 0.2ml/kgds in groups

n meglumine mg/kg body ery 24 houwere used

ur consecutivade to 0.2ml/distilled watemortality rads were o

and water inexhibited bytality in ea

drawn from72 and 96 howas immed

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mediately. Thnd those in ined for grosperiment. Tbird. Tissue n and ske

h bird and fixThese sampbedding techhickness. Tatoxylin andes. The stathologic cha

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Mortality raNo mortalityB (flunixin mwas died inmg/kg) at 56group D (flu59 hours whistart of exp(flunixin megand 69hoursmortality in 20%, 40% an

Figure-1:var

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SULTS

ed in groups 1.25 mg/kg (flunixin m

er first dosinumine, 5 mg/ne died at 75he three bir

mg/kg) wert exposure D and E w

pectively.

broiler chickef flunixin megl

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ed accordingUniversity

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ed using PRICA, USA). Malculated us

ween two met. A p<0.05 wce between

A (control) b w). One b

meglumine, ng. One bird/kg) was die5 hours after rds in groupre died at 51

(Fig. 1). was recorded

ens exposed tolumine

mpletion of pproximatelyed the signsd reluctancefelt difficultyhed aside wAffected b

with their heen forced to

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d in d at

r the p E , 63 The

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o

the y 24 s of e to y in with irds

eads and

n of

Muhammad Ramzan, et al /J. Pharm. Sci. & Res. Vol.4(2), 2012, 1748-1754

1749

affected affected mainly btreatmenremaineappearedsigns of Gross LPostmorthat diedrevealedThe mawidesprematerialand variThe preclocally etissues musclesserosal abdominserosal ventricuarticularjoints. urethra aThe liveswollen.were scaOther foshowed discolorslaughtepectoral in the bdose rateNo grosand B at

Figure-2with flunshowing (white/grmembran(arrows).

birds was dbirds beca

between 48 nt. The survid apparentlyd clinically h

f toxicity descLesions: rtem examind (1 in groupd almost simiajor gross leead depositiol (uric acid ced amount ocipitation of extensive ar(abdominal , thigh musurface, perinal fat, abd

surface of ulus, entire r surface wSmall uroli

and urethral er was friable. Varied amoattered on moour birds (2

mild to ation of k

ering at the muscles at t

birds treated e of 10 mg/ks lesions wert the time of

2: Broiler chicnixin meglumi

visceral gouray chalky mnes: pericard.

eep and rapiame comatos

and 72 houived birds iny normal. Thhealthy and cribed above

nation condup C, 2 in groilar lesions. esion was mon of a mixtrystals), whi

of fibrinous eurates variedeas present and cervica

uscles, air sicardium, epdominal wa

f liver, spleintestinal t

ithin mandiths were foopenings of e and kidneyounts of whost of the visbirds each moderate

kidneys wheend of expe

the site of injwith flunixi

kg b.w. re found in tnecropsy.

cken (group Dine at the dosut with variematerial) pre

dium, liver,

d. Prior to dse. Deaths o

urs after initin groups C, Dhe birds in gdid not man

e.

ucted on 6 coup D, 3 in g mild to sevture of whiteite debris (urexudate (Fig.d from multin the subcual region), sacs, thorac

picardium, sall serosal een, proventract, kidneiblar, hip anund in segmthe cloaca.

ys were palehite debris ansceral serosain groups Dswelling anen examineeriment. Necjection was oin meglumin

the birds of g

D, 5-week-olde rate of 5 mg

ed amounts oesent on the

gizzard, pe

eath, the occurred iation of D and E group A

nifest the

chickens group E)

vere and e chalky ric acid), 2). tifocal to utaneous pectoral

cic wall sternum, surface,

ntriculus, eys, and nd hock ments of

e-tan and nd fibrin .

D and E) nd pale ed after crosis of observed ne at the

groups A

d) treated g/kg b.w. of urates e serous eritoneum

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HistopatholoBasically, alhad similar mKidney: Ther200 µm inradiating, shtophi) that arand extendinand frequennumbers of giant cells, scattered cMultifocally,with attenuamixture of sloughed epi

Figure-3: Hisomtreated with fluurate tophi (stacell response at disrupting the aH&E, 400× . B

Liver: Theretophi surropreviously de

Figure-4:Histomwith flunixin murate crystals (tissue during tpattern of their inflammation, ncells (star). H&E

ogical Lesiol the dead b

microscopic cre are multin diameter)arp, acicularre expandingng into the adntly surroun

macrophageoccasional

cellular an, tubules ar

ated epitheliuf amorphouthelial cells a

micrograph of kunixin meglumar) have stimula

their peripheryarchitecture of Bar = 100 µm.

e are multifoounded by escribed (Fig

micrograph of meglumine 5 mg(arrows). Uratethe tissue procdeposition is v

necrotic debrisE, 600×, Bar =

ons birds in all trchanges. ifocal, variab), colorless r, crystallineg and replacindjacent corti

nded by lowes a fewer

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ated an early my (arrow). Note

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g. 4).

liver of a broig/kg (group D)s have been dicessing, howevvisible, surroun, and a few m100 µm.

reatment gro

bly-sized (upto basoph

e deposits (ung renal tubuical interstitiw to mode

multinucleas, and a rhectic debdilated (ectatually contaihilic materdebris (Fig.

of a broiler chi(group E). Sev

multinucleated gacicular urate t

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iler chicken tre, showing radiaissolved out ofver, the crystaded by heterop

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bris. tic), in a rial, 3).

cken veral giant tophi ules.

ized as

eated ating f the

alline philic giant

Muhammad Ramzan, et al /J. Pharm. Sci. & Res. Vol.4(2), 2012, 1748-1754

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Heart: Mdescribemuscle (

Figure-5:chicken trTophus foradiating heterophilcells (arro

Figure-6:chicken trSection sheterophilgiant cells Skeletal describeacute nesome maand cellmyodegchanges phagocySpleen: many urNo histoof birds

BiochemSerum collected5.29±0.9

Multifocal, raed are scat(Fig. 5).

Histomicrograpreated with flunormation is show

urate crystallic inflammatioows). H&E, 600

Histomicrogrreated with flushowing severelic infiltrates (ths (arrowhead). H

muscle: Beed there locaecrosis withacrophages, lular and kaeneration w

accompanytosis and sat The spleni

rate tophi as popathologicain group A (

mical Changuric acid cd before tre95 mg/dL,

andom, uratettered throu

ph of cardiacnixin megluminwn in a section ls surrounded on and occasio0×, Bar =200 µm

raphof skeletaunixin meglumie myodegenerhin arrows) andH&E, 400× Bar

esides urate ally extensivh large numa few multin

aryorrhectic with intra-sarnied by mtellitosis (Figic parenchympreviously dl change wa(Control).

. ges concentration

eatment were5.4±0.59

e tophi as prughout the

c muscle of ne 10 mg/kg ((star). Lesions

by macrophonal multinuclem.

al muscle of ine 10 mg/kg (ration (star) wd scattered multr = 100µm.

tophi as prve to diffuse

mbers of hetenucleated giadebris, with

rcoplasmic fmild to mg. 6). ma is scatterdescribed. s observed in

ns in the e 5.33±0.68 mg/dL, 5.

eviously cardiac

a broiler group E,). consist of

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reviously e area of erophils, ant cells, h severe floccular moderate

red with

n organs

samples mg/dL,

.42±0.53

maDiihu4cst

Fcd

Fcd

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mg/dL and 5and E, respecD and 3 in gin these valuintervals. Thhours post exuric acid con40 to 80 concentrationsurvived birdto those in co

Figure-7: Sechickens (grodifferent time

Figure-8: Sechickens (grodifferent time

Figure-9: Sechickens (grodifferent time

5.37±0.69 mctively. One group E showues in sampese birds ultixposure (Figncentrations mg/dL. Ho

ns in the ds in treatmeontrol birds.

erum uric acoup C, flunix

intervals.

erum uric acoup D, flunixintervals.

erum uric acoup E, flunixintervals.

mg/dL in groubird in grou

wed a considples collectedimately diedg. 7-9). Befo

in these birowever, ser

samples cent groups w

cid concentraxin meglumin

cid concentraxin meglumi

cid concentraxin meglumin

ups A, B, Cup C, 2 in grderable incred at subsequ

d within 48 toore death, serrds ranged frrum uric acollected fr

were compara

ations in brone 2.5 mg/kg

ations in broine 5 mg/kg

ations in brone 10 mg/kg

C, D roup ease uent o 96 rum

from acid

from able

oiler

g) at

oiler ) at

oiler

g) at

Muhammad Ramzan, et al /J. Pharm. Sci. & Res. Vol.4(2), 2012, 1748-1754

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Similarlyin samplD and E0.39±0.0mg/dL, increaseserum urSerum cranges ichanges control g

Figure-1chickens different

Figure-1chickens different

Figure-1chickens different

y, serum creles collected

E, were 0.39±07 mg/dL, 0

respectiveled in the samric acid and

creatinine coin survived were observ

group.

10: Serum cr(group C, f

time intervals

11: Serum cr(group D,

time intervals

12: Serum cr(group E, f

time intervals

eatinine concd pretreatmen±0.06 mg/dL

0.38±0.06 mly. These me birds whwere died afncentrationsbirds in tre

ved in these v

reatinine conflunixin megls.

reatinine conflunixin meg

s.

reatinine conflunixin megls.

centrations mnt in groups L, 0.39±0.03

mg/dL and 0.creatinine

hich showedfterward (Fig remained ineatment grouvalues in the

ncentrations inlumine 2.5 m

ncentrations inglumine 5 m

ncentrations inlumine 10 m

measured A, B, C,

3 mg/dL, .39±0.03

values d rise in g.10-12). n normal ups. No

e birds in

n broiler

mg/kg) at

n broiler

mg/kg) at

n broiler

mg/kg) at

PA1Urd2aA55NAc

Fitddcomt1rrfitsomd(b5esadAtudaiPadib

PretreatmentAST for g126.79±11.9U/L, 126.1±respectively. drawn befo22.26±3.54 and 23.84 UALP activity533.44±29.8536.44±37.1No significaAST, ALT acontrol group

Flunixin megits toxicity inthis drug cadiclofenac indrug causedchickens (Fiobserved witmortality oftreated with1.0mg/kg (6reported in frates of 1.25flunixin and in different bthe birds thasuch as depreof treatment.manifestationdiclofenac (6(22) observebudgerigars t5.5mg/kg boexhibited clistudy. The attributed to drug. A key obserthat died exhuric acid condie had uricappears to increase in uPossible corrand NSAIDdifferent birinduced clinbroiler chick

t serum enzgroups A, 5 U/L, 121.0±10.11 U/L

Serum ALore dosing U/L, 21.6±2

U/L in groupy in these gro7 U/L, 8 U/L and ant changes and ALP in ps.

DISC

glumine, an n broiler chian be used n animals. Td dose-depeng. 1). Compth diclofenacf 75-100% h diclofenac 6, 9). Whilfowls treated5 to 10mg/kdiclofenac e

bird species. at died exhiession, anore These signsns in vultur6, 9, 19). Coed lack of ctreated with

ody weight, inical signs difference the variation

rvation of thhibited sevencentration, c acid in nbe a causa

uric acid andrelation betw

D toxicity hrds and mamnical signs sikens (19). Al

yme activityB, C, D

07±15.57 U/L and 123LT activity

were 232.62 U/L, 2

ps A, B, C, Doups was 536

542.88±540.42±23.9were obse

birds among

CUSSION NSAID, wa

ickens to detas a safer

This study shndent morta

parable mortc in broiler cwas recordat the dos

le 33-50% d with diclofekg (19). Thiexhibit compThis study aibited severeexia and leths were similares and fowontrarily, Peclinical signs

flunixin at ta dose at wof toxicity in observat

n in species s

his study waral folds incwhile the bi

normal rangeal relationshd flunixin le

ween an increhas also bemmals. In fimilar to tholthough not m

y measured and E, w

/L, 126.21±93.8±10.12 Uin the samp.43±3.46 U

21.15±3.15 UD and E, w6.15±28.12 U±56.34 U96 respectiverved in serg treatment

as evaluated termine whetr alternativehowed that ality in brotality rates wchickens (21)ded in vultuse rate of 0

mortality wenac at the dis suggests arable morta

also showed e clinical si

hargy within ar to the clin

wls treated wereira &Werts of toxicitythe dose ratewhich the b

in the prestions might sensitivity to

as that the bcrease in serirds that did es. Thus, th

hip between ethality in biease in uric aen reportedfowls, NSAose observedmeasured in

for were 9.04 U/L, ples U/L, U/L

while U/L, U/L, vely. rum and

for ther

e to this

oiler were ). A ures 0.8-was

dose that

ality that igns 24h

nical with ther y in e of irds sent

be the

irds rum not

here an

irds. acid

d in AIDs d in the

Muhammad Ramzan, et al /J. Pharm. Sci. & Res. Vol.4(2), 2012, 1748-1754

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present study, earlier studies have shown that an increase in blood uric acid might result in acidosis that has been attributed to the development of signs reported in the present study. In fowl, cattle and goat, acidosis has been linked to CNS depression (23), weakness, reluctance to suckle, deep respiration, coma, recumbency, sudden collapse and rapid death (24). We propose that the kidney damage observed in the present study and many earlier studies (25, 26, 27, 28) may interfere with uric acid and bicarbonate excretion, resulting in the development of acidosis (20, 29) and ensuing toxicity. Postmortem examination of the birds that died exhibited severe visceral gout with excessive deposition of urates was the main postmortem lesion observed in the birds exposed to various doses of flunixin meglumine. Shultz et al. (8) found complete and highly significant association between the presence of diclofenac residues and visceral gout in dead and dying vultures collected from the wild. The findings of present study are in agreement with previous studies in which acute visceral gout with deposition of uric acid crystals on the surfaces of all visceral organs was reported in vultures died due to the diclofenac toxicity in Southeast Asia (5, 6, 30, 31). Renal and visceral gout also seen in Siberian cranes treated with flunixin meglumine (32). Necrosis of muscle tissue at the injection site was observed in this study. Similarly, severe, focally extensive pectoral muscle necrosis was reported in northern bobwhite quail injected with flunixin meglumine at dose rate of 35 mg/kg (33). Histopathological examination was conducted on the tissue samples of kidney, liver, spleen, heart and skeletal muscles from the birds treated with flunixin meglumine. Kidneys of the birds that died showed renal tubular damage, deposition of urates tophi with macrophages and heterophil infiltration, presence of amorphous, eosinophilic material in the tubular lumen. This indicates the renal tissue damage resulted from toxic effects of the drug. These results are supported by the earlier studies which indicated the renal damage and uric acid crystal deposition in the renal tissues collected from the dead or dying vultures affected with the residues of diclofenac (6, 30, 31). Similar findings were reported in experimentally treated vultures and chickens with diclofenac (6, 9, 19, 21, 34). Flunixin meglumine is responsible for developing renal papillary necrosis in horse (35). Necrosis of renal tissue was noted in dogs receiving flunixin meglumine and methoxyflurane (26). Similar observations were also

reported in quails and budgerigars treated with flunixin meglumine (22, 33). The lesions similar to those found in kidney tissue were also present in liver, heart, spleen and muscle samples. These observations are aligned with the various studies reported in literature (19, 21, 31, 33). The presence of lesions in liver persuaded us to study the enzymes that measure liver-function. In present study no significant change in ALT, AST and ALP activities were observed. These findings concur with the observations of Swan et al. (36) who also reported no clear pattern of change in ALT activity in response to diclofenac treatment in vultures. Thus, we propose that, despite microscopic damage in liver, liver function may not change in response to the flunixin dose used in chickens. Contrarily to the present observation, other studies have shown a rise in the levels of these enzymes in response to NSAID treatment in chickens (21) and dogs (26, 27). This could be attributed to the differences in the drug, dose of the drug, species and experimental design. In conclusion, this study demonstrates that flunixin, similar to diclofenac, may be toxic to chicken. We propose that a rise in the blood uric acid and creatinine may be positively correlated with the toxicity. A rise in blood uric acid and creatinine in broilers treated with diclofenac at the dose rate of 5mg/kg was reported in earlier study (21). Also the flunixin-induced kidney damage may suppress uric acid excretion in urine and results in hyperuricaemia and ensuing toxicity. The increase in serum creatinine may be the result of kidney damage (26, 27, 28). This hypothesis is supported by earlier studies using diclofenac in birds and mammal (6, 9).

ACKNOWLEDGEMENT This study was supported by the Higher Education Commission of Pakistan.

REFERENCES 1. Prakash, V.: Status of vultures in Keoladeo National Park,

Bharatpur, Rajasthan, with special reference to population crash in Gyps species. J. Bomb. Nat. Hist. Soc., 1999; 96:365–378.

2. Prakash, V., Pain, D. J., Cunningham, A. A., Donald, P. F., Prakash, N.:

3. Catastrophic collapse of Indian white-backed Gyps bengalensisand long-billed

4. Gyps indicusvulture populations. Bio. Con., 2003; 109: 381–390.

5. Gilbert, M., Virani, M. Z., Watson, R. T., Oaks, J.L., Benson, P.C., Khan, A. A., Ahmed, S., Chaudhry, J., Arshad, M., Mehmood, S., Shah, Q. A.: Breeding and mortality of Oriental white-backed vulture Gyps bengalensisin Punjab Province, Pakistan. Bird Conserv Int., 2002); 12: 311–26.

Muhammad Ramzan, et al /J. Pharm. Sci. & Res. Vol.4(2), 2012, 1748-1754

1753

6. IUCN.: IUCN Red List of Threatened Species., 2006 http://www.iucnredlist.org/

7. Pain D. J., Cunningham, A. A., Donald, P. F., Duckworth, J. W., Houston, D. C., Katzner, T., Parry-Jones, J., Poole, C., Prakash, V., Round, P., Timmins, R.: Causes and Effects of Temporospatial Declines of Gyps Vultures in Asia. Conserv Biol., 2003; 17 (3): 661–671.

8. Oaks, J. L., Gilbert, M., Virani, M. Z., Watson, R. T., Meteyer, C. U., Rideout, B., Shivaprasad, H. L., Ahmed, S., Chaudhry, M. J. I., Arshad, M., Mahmood, S., Ali, A., Khan, A. A.: Diclofenac residues as the cause of vulture population declines in Pakistan. Nature., 2004; 427, 630–633.

9. Risebrough, R.: Conservation biology: fatal medicine for vultures. Nature., 2004; 427(6975):596-8.

10. Shultz, S., Baral, H. S., Charman, S., Cunningham, A.A, Das, D., Ghalsasi, G. R.: Diclofenac poisoning is widespread in declining vulture populations across the Indian subcontinent. Proc R SocLond B., 2004; 271: S458–60.

11. Swan, G.E., Cuthbert, R., Quevedo, M., Green, R.E., Pain, D.J., Bartels, P., Cunningham, A.A., Duncan, N., Meharg, A., Oaks, J.L., Parry-Jones, J., Schultz, S., Taggart, M.A., Verdoorn, G.H., Wolter, K.: Toxicity of diclofenac in Gyps vultures., Biol. Lett., 2006a; 2: 1–4.

12. Chay, S., Woods, W. E., Nugent, T.: The pharmacology of nonsteroidal anti-inflammatory drugs in the horse: flunixin meglumine (Banamine). Equine Pract., 1982; 4:16–23.

13. Erkert, R. S., MacAllister, C. G., Payton, M. E., Clarke, C. R.: Use of force plate analysis to compare the analgesic effects of intravenous administration of phenylbutazone and flunixin meglumine in horses with navicular syndrome. Am J Vet Res., 2005; 66 (2): 284-288.

14. Kallings, P.: Nonsteroidal anti-inflammatory drugs. Vet Clin North Am Equine Pract., 1993; 9: 523–562.

15. Baskett, A., Barton, M. H., Norton, N., Anders, B. and Moore, J. N.: Effect of pentoxifylline, flunixin meglumine, and their combination on a model of endotoxemia in horses. Am J Vet Res., 1997; 58: 1291–1299.

16. Hirsch, A. C., Philipp, H., Kleemann, R.: Investigation on the efficacy of meloxicam in sows with mastitis–metritis–agalactiasyndrome. J. Vet. Pharmacol. Therap., 2003; 26: 355–360.

17. Drillich, M., Voigt, D., Forderung, D., and Heuwieser, W.:Treatment of acute puerperal metritis with flunixin meglumine in addition to antibiotic treatment. J. Dairy Sci., 2007; 90: 3758–3763.

18. Lockwood, P. W., Johnson, J. C., Katz, T. L.: Clinical efficacy of flunixin, carprofen and ketoprofen as adjuncts to the antibacterial treatment of bovine respiratory disease. Vet Rec., 2003; 152: 392-394.

19. Baert, K., De Backer, P.: Disposition of sodium salicylate, flunixin and meloxicam after intravenous administration in broiler chickens. J. Vet. Pharmacol. Ther., 2002; 25(6): 449–453.

20. Baert, K., De Backer, P.: Comparative pharmacokinetics of three non-steroidal anti-inflammatory drugs in five bird species. Comp. Biochem. Physiol. C ToxicolPharmacol., 2003; 134 (1): 25–33.

21. Naidoo, V., Duncan, N. ,Bekker, L., Swan, G.: Validating the domestic fowl as a model to investigate the pathophysiology of diclofenac in Gyps vultures. Environ. Toxicol. Pharmacol., 2007; 3: 260-266.

22. Speer, B. L.: Diseases of the urogenital system. In Avian Medicine and Surgery. (Eds) Altman, R. B., Clubb, S. L., Dorrestein, G. M., Quesenberry, K. Philadelphia, W. B. Saunders., 1997; 625-644.

23. Reddy, P. N. C., Anjaneyula, Y., Sivasankari, B., Rao, A. K.: Comparative toxicity studies in birds using nimesulide and

diclofenac sodium. Environ. Toxicol. Pharmacol., 2006; 22: 142–147.

24. Pereira, M. E., Werther, K.: Evaluation of the renal effects of flunixin meglumine, ketoprofen and meloxicam in budgerigars (Melopsittacusundulatus). Vet Rec., 2007; 160(24): 844-6.

25. Blood, D.C., Radostits, O.M.: Veterinary Medicine. A textbook of Diseases of Cattle, Sheep, Pigs, Goats and Horses. BailliereTindall, London., 1989; 64-66.

26. Radostits, O. M., Gay, C. C., Hinchcliff, K. W., Constable, P. D.: Disturbances of free water, electrolytes and acid-base balance. In Veterinary Medicine. 10th Ed. Saunders, Elsevier., 2007; 82-86.

27. Elwood, C., Boswood, A., Simpson, K., Carmichael, S.: Renal failure after flunixin meglumine administration. Vet Rec., 1992; 130(26): 582-583.

28. Mathews, K. A., Doherty, T., Dyson, D. H., Wilcock, B., Valliant. A.: Nephrotoxicity in dogs associated with methoxyflurane anesthesia and flunixin meglumine analgesia. Can Vet J., 1990; 31: 766-771.

29. Mathews, K. A., Paley, D. M., Foster, R. F., Valliant, A., Young, S. S.: A comparison of ketorolac with flunixin, butorphanol, and oxymorphone in controlling postoperative pain in dogs. Can Vet J., 1996; 37: 557-567.

30. Murray, M. D. &Brater, D. C.: Renal toxicity of the nonsteroidal anti-inflammatory drugs. Annu. Rev. Pharmacol. Toxicol., 1993; 33: 435–465.

31. Seifter, J. L.: Acid–base disorders. In: Arend,W. P., Drazen, J. H., Ill, G. N., Riggs, R. C., Owell, D. W., Cheld, W .M. (Eds.), Cecil Textbook of Medicine. W.B. Saunders Company, Philadelphia., 2004; 688–695.

32. Cunningham, A. A., Prakash, V., Pain, D., Ghalsasi, G. R., Wells, G. A. H., Kolte, G. N., Nighor, P., Goudar, M. S., Kshirsagar, S., and Rahmani, A.: Indian vultures: Victims of an infectious disease epidemic? Animal Conservation., 2003; 6: 189–197.

33. Mishra, S. K., Prasad, G., Minakshi, Malik, Y., Mahajan, N. K. and Prakash, V.: Vulture mortality: pathological and microbiological investigations. Indian J Anim Sci., 2002; 72: 283-286.

34. Clyde, V. L., Murphy, J.: Avian analgesia. In: Fowler, M. E., Miller, R. E. (Eds.), Avian Medicine. Zoo &Wild Animal Medicine: Current Theory, vol. 4. W.B. Saunders, Philadelphia., 1999; 309–314.

35. Klein, P. N., Charmatz, K. and Langenberg, J.: The effect of flunixin meglumine (Banamine) on the renal function in northern bobwhite (Colinusvirginianus): An avian model. Proceedings of the American Association of Zoo Veterinarians and Association of Reptilian and Amphibian Veterinarians Conference, Pittsburgh, Pennsylvania, R. E. Junge (Ed.). American Association of Zoo Veterinarians, Philadelphia, Pennsylvania., 1994; 128–131.

36. Meteyer, C. U., Rideout, B. A., Gilbert, M., Shivaprasad, H. L. & Oaks, J. L.: Pathology and proposed pathophysiology of diclofenac poisoning in free-flying and experimentally-exposed oriental white-backed vultures (Gyps bengalensis). J. Wildl. Dis., 2005; 41: 4.

37. Faulkner, L. W., Erb, H. N. and King, J. M.: Renal Papillary Necrosis in Equines.Bull Environ. Contam. Toxicol., 1984; 33: 379-381.

38. Swan, G., Naidoo,V., Cuthbert, R., Green, R.E., Pain, D.J., Swarup, D., Prakash, V., Taggart, M., Bekker, L., Das, D., Diekmann, J., Diekmann, M., Killian, E., Meharg, A., Patra, R.C., Saini, M., Wolter, K., Removing the threat of diclofenac to critically endangered Asian vultures. PLoS Biol., 2006b; 4: 395–402.

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